DNA甲基转移酶活性的高灵敏度检测及其荧光相关光谱与聚苯乙烯聚合物点的偶联剂筛选,Analyst

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DNA甲基转移酶活性的高灵敏度检测及其荧光相关光谱与聚苯乙烯聚合物点的偶联剂筛选
Analyst ( IF 3.6 ) Pub Date : 2021-4-9 , DOI: 10.1039/d0an02362k
Yuyang Huang _{1,

2,

3,

4,

5} , Liyun Deng _{1,

2,

3,

4,

5} , Di Su _{1,

2,

3,

4,

5} , Xiangyi Huang _{1,

2,

3,

4,

5} , Jicun Ren _{1,

2,

3,

4,

5}

Affiliation

DNA甲基化是表观遗传学的关键部分，在维持正常的细胞功能，基因印记和人类肿瘤发生中起着至关重要的作用。因此，开发一种测定DNA甲基转移酶（MTase）活性的灵敏方法非常重要。在这里，我们提出了一种基于单分子荧光相关光谱（FCS）和聚苯乙烯聚合物点（PS Pdots）的简单而灵敏的方法，用于定量检测DNA腺嘌呤甲基化（Dam）MTase活性及其在均相溶液中无需分离的抑制剂筛选。其原理是基于的特征扩散时间的测量（τ _dFCS分析未甲基化和甲基化的DNA荧光探针）。通过分别在5'和3'末端双标记荧光团Alexa Fluor 488（Alexa 488）和生物素，使用包含5'-GATC-3'序列的发夹DNA探针。Dam MTase催化5'-GATC-3'序列的甲基化，DpnI切割5'-G-Am-TC-3'的序列。链亲和素缀合的P点PS使用了未经甲基化的DNA探针进行反应，以进一步增加差τ _d甲基化和未甲基化DNA的Alexa488的探针之间的值。我们使用FCS方法来测量τ _d DNA-Alexa488的探针的值，并进一步获得坝转移酶的活性。据发现，τ _d甲基化DNA探针的值在0.025 U mL ^-1至3 U mL ^-1范围内与Dam MTase浓度的对数负相关。检出限低至0.025 U mL ^-1。此外，我们评估了药物相关的DNA甲基化的抑制作用，半数最大抑制浓度（IC ₅₀）值与先前的研究一致。结果表明，我们提出的方法将成为确定大坝MTase活性和抑制剂筛选的有前途的平台。

"点击查看英文标题和摘要"

Highly sensitive detection of DNA methyltransferase activity and its inhibitor screening by coupling fluorescence correlation spectroscopy with polystyrene polymer dots

DNA methylation is a critical part of epigenetics and plays a vital role in maintaining normal cell function, genetic imprinting, and human tumorigenesis. Thus, it is important to develop a sensitive method for the determination of DNA methyltransferase (MTase) activity. Here, we present a simple and sensitive method based on single molecule fluorescence correlation spectroscopy (FCS) and polystyrene polymer dots (PS Pdots) for the quantitative detection of DNA adenine methylation (Dam) MTase activity and its inhibitor screening in homogeneous solution without separation. Its principle is based on the measurement of the characteristic diffusion time (τ_D) of unmethylated and methylated DNA-fluorescent probes by FCS. A hairpin DNA probe including the 5′-GATC-3′ sequence is used by doubly labelling fluorophore Alexa Fluor 488 (Alexa 488) and biotin at the 5′- and 3′-terminus, respectively. Dam MTase catalyzed the methylation of the sequence of 5′-GATC-3′, and DpnI cleaved the sequence of 5′-G-Am-TC-3′. Streptavidin conjugated PS Pdots were used to react with DNA probes without methylation to further increase the difference in τ_D values between methylated and unmethylated DNA-Alexa 488 probes. We used the FCS method to measure the τ_D values of DNA-Alexa 488 probes and further obtained the activity of Dam MTase. It is found that the τ_D value of the methylated DNA probe is negatively correlated with the logarithm of Dam MTase concentration in the range from 0.025 U mL⁻¹ to 3 U mL⁻¹. The detection limit is as low as 0.025 U mL⁻¹. Furthermore, we evaluated the inhibition effect of drug-related DNA methylation and the half-maximal inhibitory concentration (IC₅₀) value is consistent with a previous study. The results demonstrated that our proposed method will become a promising platform for the determination of Dam MTase activity and inhibitor screening.

更新日期：2021-04-30

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